The transient introduction of proteins of interest into a target cell is a major challenge, with many applications in basic research, applied science and in the therapeutic field.
For decades, introduction of a protein of interest into a target cell has been accomplished indirectly, by the introduction of genetic material encoding said protein of interest, known as transfection.
Many transfection techniques have been developed over the years, such as calcium phosphate transfection, electroporation and lipofection.
However, these techniques have several inconvenients that many limit their use. The introduction of a gene encoding a protein of interest into a target cell relies on the transcription and translation machinery of the target cell. The expression of the desired protein is thus slow, and several undesirable effects can be observed (toxicity of the transfection reagent itself, activation of the interferon response etc.).
Moreover, certain cell types, typically non-dividing cells such as neurons, are poorly transfectable. Other gene transfer techniques known as transduction have been developed more recently in order to circumvent this drawback. Transduction relies on the use of a viral vector to transfer genetic material to a target cell. Retrovirus-based systems are often used to this effect. However, these technologies suffer from several drawbacks. First, there is a sanitary risk associated with the use of retroviral vector, even though said retroviral vectors have been inactivated. Second, production of retroviral particles is very tedious and time-consuming. Indeed, it involves the production of large amounts of 4 or 5 different plasmids which encode the various proteins necessary for the formation of the viral vector particles. Finally, the effects observed in the target cell are not immediate. Typically, the protein of interest is expressed in the target cell after a delay of at least 24 hours, usually 48 to 72 hours, during which the transcription and translation take place.
Previous works described the unexpected transfer of reporter proteins by by-standard agents co-produced with retroviral vectors (Liu et al., Journal of virology 70, 2497-2502 (1996); Gallardo et al., Blood 90, 952-957 (1997). For example, these authors described the ability of their viral preparation to deliver β-Galactosidase in target cells known to be non-permissive to retrovirus mediated gene transfer. This undesirable transduction artefact termed as pseudotransduction led to a transient expression of the transferred protein in the treated cell while retroviral transduction ensures a stable long term expression of the delivered transgene. Interestingly these authors used retroviral vectors pseudotyped with the G protein of the Vesicular Stomatitis Virus (VSV-G) and observed that pseudotransduction occurred specifically with concentrated VSV-G coated retroviral particles. However, these studies provided no conclusive evidence of the mechanism underlying this protein-mediated pseudotransduction and it was thought that the transferred protein was most likely integrated into the viral envelope (Schnell et al., PNAS, 1996, 93, 11359-11365). This pseudotransduction was therefore thought to be limited to membrane proteins. Moreover, it was believed to be non-dissociable from gene-mediated retroviral transduction.
In order to obtain a specific, time-controlled delivery of a protein of interest into a target cell, another available technique is microinjection into the target cell. However, this technique requires the obtaining of the protein of interest prior to microinjection. Peptides can be obtained by chemical synthesis, whereas bigger proteins will require specific production and purification procedures. Thus, these methods are costly and poorly amenable to large-scale uses.
Other studies have focused on the delivery of proteins by conjugating them with a short peptide that mediates protein transduction, such as HIV tat and poly-arginine. However, these techniques are also difficult to implement on a large-scale basis, since they rely on recombinant proteins.
Thus, there is still a need in the art for a fast, safe and efficient method for delivering a protein of interest into a target cell.